Project Summary/Abstract: The development of fatal paralysis in amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the central nervous system (CNS). Inhibiting the persistent and toxic neuroinflammation around motor neurons is a promising pharmacological strategy to prevent disease progression. Conventional anti-inflammatory drugs have limited CNS activity and have not been effective in ALS to date. Regulatory T cells (Tregs) are a subset of lymphocytes with inherent anti-inflammatory activity, are capable of penetration into the CNS, and higher numbers of Tregs are associated with slower disease progression in ALS patients. The overall objective of this research project is to develop a cell therapy consisting of genetically engineered, target-directed Tregs designed to inhibit neuroinflammation in ALS patients. The goal of this proposal is to engineer human Tregs with chimeric antigen receptors (also called CARs) that will bind and induce their regulatory function when in contact with their target proteins that are known to be abnormally expressed at areas of motor neuron loss and neuroinflammation in ALS patients. Mutant SOD1 (mutSOD1) is located both intracellularly and in the extracellular space near diseased neurons. In this project, CAR-expressing human Tregs specific for human mutSOD1 will be developed and assessed for antigen-specific neuroprotective and anti-inflammatory activities in vitro. These anti-mutSOD1 CAR Tregs will be evaluated in two in vivo models of ALS where mutSOD1 is expressed. The ability of the CAR Tregs to localize to sites of neuroinflammation, decrease local inflammation, and increase overall survival will be evaluated. The expected outcome is that CAR Tregs will generate anti-inflammatory cytokines, inhibit local inflammation, and protect against microglial/macrophage-mediated motor neuron death resulting in decreased progression of disease and increased survival in these ALS models. A potential therapeutic breakthrough with therapeutic CAR Tregs would have a major impact on patients, their families, and clinical management of ALS.